Collection and preparation of specimens
Samples of the West African Mud Creeper, T. fuscatus var radula (Linnaeus, 1758) were collected by scooping from the waterbed at low tides from the Abule-Agege Creek in Southwest Nigeria, between January and December 2019. The site lies between latitudes 6° 26′–37′ N and longitude 3° 23′–4° 20′ E (Moruf et al. 2018). The shores of the creek are lined in undeveloped areas by mangrove swamps. An elaborate description of the study area was provided by Moruf and Ojetayo (2017).
To extract adhering sediments and organic particles, fresh samples were washed with purified water several times. The samples were transported in a 10-L bucket to the laboratory and processed within 4 h of the collection. Using a bench vice, the shell was split and the viscera removed. The edible parts were used for all analyses. These samples were divided into two groups, with 10 samples of muscles in each group, with mean flesh weight of 2.76 ± 1.05 g. The first group (raw sample) was dried directly in the oven at 70 °C for 48 h and the second group was cooked in 200 ml of distilled water for around 60 min by steaming using an automatic cooker (2000 W, Tefal, Zahran). At 70 °C, the steamed samples were also dried for 48 h. All dried specimens were weighed using a digital balance and homogenized using a kitchen blender.
Laboratory analysis
Determination of biochemical profiles
According to the methods of AOAC (2000), water content, crude protein, crude fat, crude fiber, ash, and carbohydrate content were determined. By means of the already confirmed amount of proteins and fat, the energy content of the edible part of the organism was calculated. The energy values (KJ/100 g) of the specimen were estimated, multiplying the amount of proteins (%) by factor 17.16 and multiplying the amount of fat (%) by factor 38.96 and then calculating the number of the two already determined values (Saveski et al. 2017).
Using a flame photometer (model 405, Corning, UK), some macro minerals (sodium and potassium) were determined, while other minerals were determined by the Atomic Absorption Spectrophotometer (Perkin & Elmer model 403, USA) as defined by Gokoglu and Yerlikaya (2003). Mineral Safety Index was calculated from Eq. 1 as:
$${\text{MSI}} = \left[ {{\text{TMSI }}\left( {{\text{Standard}}} \right)/{\text{RAI}}} \right].{\text{ Rr}},$$
(1)
where MSI—Mineral Safety Index; TMSI—Tabulated MSI; RAI—Recommended Adult Intake; and Rr—Researh results.
Using Liebermann–Burchard reagents, the cholesterol content was measured. The Liebermann–Burchard reagent was prepared with 2 ml of glacial acetic acid and 0.2 mL of concentrated sulfuric acid and then coated with aluminum foil. The chemicals were purchased from a commercial source (Fisher Scientific, USA) and used as received. Gas chromatography of methyl esters was used to separate and determine the volatile content of fat derived from samples (AOAC 2000). The GC analyses were performed on 7890A gas chromatography system (Agilent Technologies, California, USA) equipped with flame ionization detector and splitless injector (1 μL). Injector and detector temperature were set at 270 °C and 280 °C, respectively. In the determination of free fatty acid (FFA) as Oleic, one (1) g of the test sample was poured into a conical flask with 20 ml of solvent mixture (1:1 v / v ethanol: diethyl ether) and 0.2 ml of phenolphthalein indicator solution. The mixture was then titrated until the pink color appears and persists for at least 10 s when shaking with 0.1 N KOH. The % FFA was calculated from Eq. 2 as:
$$\% {\text{FFA}} \left( {{\text{as}}\;{\text{ Oleic}}\;{\text{ Acid}}} \right) = \frac{{\left( {V {-} B} \right) \times N \times 28.21}}{W}$$
(2)
where V volume of titrant (KOH) consumed for sample, B volume of titrant consumed for blank, N normality of titrant (KOH), W weight of sample.
Determination of non-enzymatic antioxidants and functional properties
Thiobarbituric acid (TBA) was colorimetrically determined in flesh samples of T. fuscatus var radula, as described by Torres Arreola et al. (2007). 10 g of minced flesh was macerated for 2 min with 50 ml of distilled water, washed with 47.5 ml of distilled water into a distillation flask with addition of 2.5 ml of hydrochloric acid (4 N). A volume of 50 ml distillate was collected from which 5 ml of distillate was pipetted into a glass tube and mixed with 5 ml of TBA reagent. After cooling the mixture, which was heated for 35 min in a boiling water bath, the optical density was measured against the blank at a wavelength of 538 nm. A method based on the spectrometric quotation of the pink complex produced the after reaction of one malondialdhyde molecule (MDA) with two TBA molecules. The TBA was calculated from Eq. 3 as:
$${\text{TBA}} \left( {{\text{Distillation}}} \right) = \frac{C. V}{{{\text{Ms}}}}$$
(3)
where C is the concentration of MDA (\(\pi\) M) as read from the calibration curve; V is the volume of the distillate (ml); and Ms is the mass of the sample (g).
By modifying a protocol described by Phenomenex (2011), the chromatographic method for evaluating ascorbic acid in fresh meat preparations was employed. 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) was determined as described by Farvin et al. (2010), where in 20% ethanol, 2 mL of 0.1 mM DPPH was combined with 2 mL of 50 mg/mL sample concentration. By substituting samples with 2 mL of 6.25% ethanol, Blank was prepared. The sample was incubated with the blanks for 30 min in the dark. The absorbance was read at 520 nm using the JASCO UV–VIS spectrometer after the incubation time. The proportion of radical scavenging activity was calculated from Eq. 4 as:
$${\text{Radical }}\;{\text{scavenging}} \;{\text{activity}} \left( \% \right) = \frac{{{\text{A}}\;{\text{ blank}} - {\text{A}} \;{\text{sample}}}}{{{\text{A}} \;{\text{blank}}}} \times \, 100$$
(4)
With regard to functional properties, water absorption capacity (WAC) and oil absorption capacity (OAC) were determined following the method described by Brishti et al. (2017). The 0.25 g of specimen was mixed with 5 ml distilled water or oil in pre-weighed centrifuge tube for 30 s using a vortex. Then, specimen was allowed to stand at room temperature (20–25 °C) for 15 min and centrifuged at 3000 rpm for 15 min. After centrifugation, the supernatant was decanted, and the centrifuge tubes + precipitate were reweighed. The WAC and OAC were expressed as grams of water/oil absorbed per gram of the sample. The WAC and OAC were calculated by using the Eq. 5 as:
$${\text{WAC}}\;{\text{ or}}\;{\text{ OAC}} \left( {\text{g/g}} \right) = \frac{W2 }{{W1}}$$
(5)
where W1 = weight of the dry sample (g); W2 = weight of precipitate + centrifuge tube (g).
The modified methods reported by Souissi et al. (2007) were used to determine the emulsion stability. The foam formation and the foam stability were determined by optical measurements. The foams were produced with a homogenizer for 2 min at 17 500 rpm, in 3 ml of solution (50 mM Tris-HCl—0.5 M NaCl, pH 7.5), which contained 1.5% protein. The initial height of the solution and the foam height were recorded at intervals of 0, 2, 10, 20 and 30 min, using a caliper. The foaming capacity was expressed as the proportion of foam height at 0 min to solution height. The foaming stability (FS) was conveyed by the percentage of foam height at some time to 0 min. The measurement of the height was rapid and accurate to three digits after the decimal point.
Statistical analysis
Data obtained was subjected to analysis of variance (ANOVA) using SPSS (Statistical Package for Social Sciences) version 24.0.0 statistical software. Statistically significant differences were identified at p < 0.05.
